Single-domain antigen-binding proteins that bind mammalian igg

ABSTRACT

The present application relates to antigen-binding proteins that are capable of binding to mammalian IgG. The frame-work regions of the antigen-binding proteins of the application preferably correspond to those of antibodies naturally that are devoid of light chains as may e.g. be found in camelids. The application further relates to nucleic acids that encode such antigen-binding proteins, to immunoadsorbent materials that comprise such proteins, to the uses of such immunoadsorbent materials for the purification of mammalian IgG antibodies and for therapeutic apheresis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 12/669,034, filed Mar. 30, 2010, which is a U.S. National PhaseApplication of International Application No. PCT/NL2008/050460, filedJul. 8, 2008, which claims the benefit of U.S. Provisional ApplicationNo. 60/949,557, filed Jul. 13, 2007; and European Patent Application No.07112456.4, filed Jul. 13, 2007; and European Patent Application No.07119634.9, filed Oct. 30, 2007, which disclosures are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of biochemistry, inparticular immunoglobulin purification and antibody technology. Theinvention relates to amino acid sequences that are capable of binding tomammalian IgG; to proteins and polypeptides comprising or essentiallyconsisting of such amino acid sequences; to nucleic acids that encodesuch amino acid sequences, proteins or polypeptides; to immunoadsorbentmaterials that comprise such proteins and polypeptides; and to uses ofsuch immunoadsorbent materials for the purification of mammalian IgGantibodies.

BACKGROUND OF THE INVENTION

Efficient, rapid, save and cost efficient purification of mammalian IgGantibodies, in particular human and/or humanized IgG antibodies is amuch studied problem in the art. With the advent of new antibody basedmedicaments, purification of IgG becomes a more and more critical andcostly step in the production of antibody based medicaments, requiring ahigh degree of purity. In addition, such antibodies must retain bindingaffinity and biological activities like effector functions.

For the purification of mammalian IgG antibodies, in particular humanIgG or humanized IgG antibodies, commonly used purification methodscomprise the use of classical biochemical separation and purificationtechniques such as anion/kation exchange, size-exclusion/gelfiltration,precipitations and use of specific affinity ligands. Commonly usedligands are bacterially derived proteins, Protein-A and Protein-G.Alternatively, Protein L can be used, but only for those immunoglobulinscomprising a kappa light chain since Protein L does not bind lambdalight chains.

Protein-A is a bacterial surface protein expressed by Staphylococcusaureus. Protein-A primarily recognizes a common site at the interfacebetween C_(H)2 and C_(H)3 domains on the Fc part of human IgG1, IgG2 andIgG4 antibodies (Fcγ). In addition, Protein-A also shows binding to 12%of mouse and 50% of human V_(H) domains (human V_(H)-III subclass).Although these latter interactions have a lower affinity (±200 nM forV_(H) compared to <1 nM for e.g. human IgG1) Protein-A can be used forpurification of Fab- and (sc)Fv fragments (independent of the Igisotype). Protein-A, like Protein L acts as a superantigen on human Blymphocytes, probably induced by its V_(H)-III reactivity. Therefore, ifthe purified IgG antibodies are intended for therapeutic usage, a majorsafety concern is the possible presence of Protein-A in the purifiedtherapeutic product as a result from the unintended detachment ofProtein-A from its support material during the purification process(Protein-A leakage). Numerous publications link Protein-A with toxicityand mitogenicity in animal models and humans (see, for example,Bensinger et al., J. Biol. Resp. Modif. 3, 347, 1984; Messerschmidt etal., J. Biol. Resp. Modif. 3, 325, 1984; Terman and Bertram, Eur. J.Cancer Clin. Oncol. 21, 1115; 1985; and Ventura et al., Hortobagyl.Cancer Treat Rep. 71, 411, 1987).

Furthermore, co-binding of Protein-A to human V_(H)-III domains is themain reason for causing elution pH differences in affinitychromatography amongst several IgG antibodies. Such differences are notdesirable because it causes a lack of consistency in purificationprocedures among different monoclonal antibodies (Mabs). Furthermore,tightly bound IgG Mabs, due to co-binding of Protein-A to human VH-III,often require a lower pH value of the eluents in order to obtainefficient recoveries.

Protein-G is a bacterial surface protein expressed by group C and Gstreptococci. Like Protein-A, Protein-G also recognizes a common site atthe interface between C_(H)2 and C_(H)3 domains on the Fc part of humanIgG1, IgG2, IgG3 and IgG4 antibodies (Fcγ). Compared to Protein-A, abroader range of IgG species can be recognized. In addition, Protein-Gshows binding to the Fab portion of IgG antibodies through binding tothe C_(H)1 domain of IgG. Binding affinity towards C_(H)1 (±200 nM) isagain significantly lower compared to its epitope on the Fc part.Although Protein-G has a wider reactivity profile than Protein-A, thebinding of antibodies to Protein-G is often stronger, making elution andcomplete antibody recovery more difficult.

The most commonly used ligand for affinity purification of humanimmunoglobulins, in particular IgG's, for large-scale processapplications is Protein-A. However, protein-A lacks the capability ofbinding to human antibodies of the IgG3 subclass. In addition, Protein-Aand G strongly bind to the CH2-CH3 interface on the Fc portion of IgGantibodies. Experimental data indicate that induced fit occurs, whichmay explain the harsh conditions required for elution. These harshconditions may affect the conformation of the binding sites, therebyaltering the immune function of purified IgG antibodies (P. Gagnon,1996, in Purification tools for monoclonal antibodies, published byValidated Biosystems, Inc 5800N). X-ray crystallographic measurementshave shown that through binding to Protein-A, the CH2 domains can bedisplaced longitudinally towards the CH3 domains, which finally causespartial rotation and destabilization of the carbohydrate region betweenthe CH2 domains. The distortion interferes with subsequentprotein-protein interactions that are required for the IgG to exert itseffector functions. Aside from the consequences of harsh elutionconditions (especially for Protein-G) on the antigen bindingcapabilities, these secondary effects sometimes interfere with or alterantibody effector functions and increased susceptibility ofimmunoglobulins to proteolysis. Loss of effector functions, caused bydenaturation, altered folding and chemical modifications that ariseduring purification steps, are highly undesirable if the human orhumanized antibodies are to be used for therapeutic purposes. Inparticular, reduction of intra- and inter-molecular sulphur bridges isoften a problem that arises during purification and storage.

As alternative to human IgG binding proteins like Protein-A and G,several mouse monoclonal antibodies (Mabs) have been described inliterature that are capable of binding to the Fc domain of human IgGantibodies. (Nelson P N, et al. Characterisation of anti-IgG monoclonalantibody A57H by epitope mapping. Biochem Soc Trans 1997; 25:373.)

Some common Fc epitopes have been identified and a number of examplesare listed below: Mabs G7C, JD312 have a binding epitope on CH2, aminoacids 290-KPREE-294. Mabs PNF69C, PNF110A, PNF211C, have a bindingepitope on CH2-CH3, AA: 338-KAKGQPR-344. Mab A57H shows binding epitopeon CH3, AA 380-EWESNGQPE-388. A problem associated with the use of mousemonoclonals, or monoclonals from other non-human species, is the releaseof Mabs from the matrix which causes contamination in the purifiedpreparations that is difficult to remove. Furthermore, monoclonalantibodies and functional fragments thereof (Fab, Fab2) are easilydenatured and S—S bridges, keeping the 3D structure of the molecule andthe heavy and light chain aligned, are easily disrupted, in particularunder harsh elution conditions that are oftentimes required for releaseof column bound human IgG's. Due to the vulnerability of the affinityligands the capacity of the column is rapidly reduced, and columns havea very limited re-use capacity after elution and are unsuitable forcontinuous operation.

Instead of (sc)Fv fragments as described in EP-A-434317, antibodyfragments derived from antibodies naturally devoid of light chains (VHH)as described in WO2006/059904 can also be used to generate immunosorbentmaterials for the purification of human IgG antibodies. Advantage of useof these VHH fragments are that they are single domain peptides, whichare exceptionally stable even at higher temperatures. Furthermore,VHH's, are small and easily produced in cost-efficient host organismssuch as Saccharomyces cerevisiae. In addition, due to the sequencesimilarity between these VHH fragments and the human V_(H)-III domainfamily, immunogenecity is expected to be very low compared to bacterialsurface proteins like Protein-A and G. These antibodies are described inmore detail in EP-A-656946.

However, the amino acid sequences as described in WO2006/059904 relateto VHH fragments that bind to the light chain of human antibodies ofeither the kappa or lambda isotype, and as such do not enable selectivepurification of antibodies of the IgG isotype only.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. In FIG. 1 the residual dynamic binding capacities (DBCs) aftercycles with 0.1 M NaOH (open diamonds) and 0.2 M NaOH (open squares) arepresented.

DESCRIPTION OF THE INVENTION

We have found a new class antigen-binding proteins that are useful forincorporation into and/or attachment to immunoadsorbent materials forthe selective purification of mammalian IgG antibodies, including humanIgG antibodies, through binding of an epitope that is present in the Fcdomain of such IgG antibodies.

In a first aspect, the present invention relates to an antigen-bindingprotein that specifically binds to a mammalian IgG. Preferably, theantigen-binding protein comprising an amino acid sequence that comprises4 framework regions, FR1 to FR4, and 3 complementarity determiningregions, CDR1 to CDR3, that are operably linked in the orderFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein: a) the CDR1 has an amino acidsequence selected from the group consisting of SEQ ID No's: 1-49 or anamino acid sequence that differs from SEQ ID No's: 1-49 in one or two ofthe amino acid residues; b) the CDR2 has an amino acid sequence havingat least 80, 85, 90, 95, 98% sequence identity with an amino acidsequence selected from the group consisting of SEQ ID No's: 50-98; and,c) CDR3 is an amino acid sequence having at least 80, 85, 90, 95, 98%sequence identity with an amino acid sequence selected from the groupconsisting of SEQ ID No's: 99-147; and, wherein each of the frameworkregions has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%amino acid identity with the framework amino acid sequence of any one ofSEQ ID No's: 148-196.

In a preferred embodiment, the antigen binding proteins of the inventionare antibodies, more preferably such antibodies or fragments thereof arederived from antibodies naturally devoid of light chains. Antibodiesnaturally devoid of light chains may be obtained e.g. by immunisation ofcamelids (e.g. llama's) or sharks (see further below). These antibodiescomprise heavy chains only and are devoid of light chains. The advantageof use of such single domain heavy chain antibodies is that they areexceptionally stable even at higher temperatures, small and are easilyproduced in host organisms such as Saccharomyces cerevisiae. Thus, anantigen-binding protein of the invention preferably comprises animmunoglobulin-derived variable domain that comprises a complete antigenbinding site for an epitope on a target molecule in a single polypeptidechain. Such antigen-binding proteins specifically include but are notlimited to:

1) antibodies obtainable from camelids and sharks that consist of onlyheavy chains and that are naturally devoid of light chains;

2) variable domains of the antibodies defined in 1), usually referred toas VHH domains;

3) engineered forms of the antibodies defined in 1) or domains in 2)such as e.g. “camelidised” antibodies in which frame work sequences of acamelid (or shark) VHH domain are grafted with CDRs obtained from othersources;

4) engineered forms of immunoglobuline-like variable domains in whichframe works sequences from a variety of immunoglobuline-like moleculesare combined with CDRs specific for a given target molecule as e.g.described in WO 04/108749.

In a preferred antigen-binding protein of the invention, the singlepolypeptide chain of the variable domain that comprises the fullantigen-binding capacity preferably has an amino acid sequence andstructure that can be considered to be comprised of four frameworkregions or “FR's”, which are referred to in the art and herein as“Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as“Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. Theseframework regions and complementary determining regions are preferablyare operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (fromamino terminus to carboxy terminus).

The total number of amino acid residues in the variable domain with fullantigen-binding capacity can be in the region of 110-135, and preferablyis in the region of 115-129. However, a variable domain with fullantigen-binding capacity in accordance with the invention is notparticularly limited as to its length and/or size, as the domain meetsthe further functional requirements outlined herein and/or is suitablefor the purposes described herein. The amino acid residues of a variabledomain with full antigen-binding capacity are numbered according to thegeneral numbering for VH domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to VHH domains fromCamelids by Riechmann and Muyldermans (1999, J. Immunol. Methods 231(1-2): 25-38, see for example FIG. 2 of said reference) and by Harmsenet al. (2000, Molecular Immunology 37: 579-590, see for example FIG. 1of said reference).

According to this numbering, in a variable domain with fullantigen-binding capacity: FR1 comprises the amino acid residues atpositions 1-25; CDR1 comprises the amino acid residues at positions26-35; FR2 comprises the amino acids at positions 36-49; CDR2 comprisesthe amino acid residues at positions 50-64; FR3 comprises the amino acidresidues at positions 65-94; CDR3 comprises the amino acid residues atpositions 95-102; and, finally, FR4 comprises the amino acid residues atpositions 103-113.

In this respect, it should be noted that—as is well known in the art forVH domains and for VHH domains—the total number of amino acid residuesin each of the CDR's may vary and may not correspond to the total numberof amino acid residues indicated by the Kabat numbering. However, basedon the conserved amino acids of the frame work region a skilled personwill be able to align the respective frame work and complementaritydetermining regions in accordance with the Kabat definitions for thosevariable domains with full antigen-binding capacity that have a lengthother than 113 amino acids. Examples thereof are given in the definitionof the complementarity determining regions in the amino acid sequencesof IgG-Fc 1-49 herein. Alternative methods for numbering the amino acidresidues of VH domains, which methods can also be applied in ananalogous manner to VHH domains from Camelids and to variable domainswith full antigen-binding capacity, are the method described by Chothiaet al. (Nature 342, 877-883 (1989)), the so-called “AbM definition” andthe so-called “contact definition”, or the IMGT numbering system(Lefranc et al., 1999, Nucl. Acids Res. 27: 209-212).

In a preferred antigen-binding protein of the invention, the frame workamino acid sequence of a variable domain with full antigen-bindingcapacity preferably has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 100% amino acid identity with the frame work amino acid sequence ofany one of SEQ ID No's: 148-196.

More preferably, the amino acid residues that are present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of the single polypeptide chain of the variable domain that comprisesthe full antigen-binding capacity preferably are as indicated in Tables1 to 4 for FR1, FR2, FR3 and FR4. Thereby preferably the frame workamino acid residues of a variable domain with full antigen-bindingcapacity are chosen from the non-limiting residues in Tables 1 to 4 thatcan be present at each position (according to the Kabat numbering) ofthe FR1, FR2, FR3 and FR4 of naturally occurring Camelid VHH domains(data was taken from patent WO 2006/040153 PCT/EP2005/011018). Morepreferably, however, the frame work amino acid residues of a variabledomain with full antigen-binding capacity are chosen from the amino acidresidues in Tables 1 to 4 that are present at each position (accordingto the Kabat numbering) of the FR1, FR2, FR3 and FR4 of the amino acidsequences of any one of SEQ ID No's: 148-196, of antigen-bindingproteins that specifically bind a mammalian IgG. For each position, theamino acid residue that most frequently occurs at each position isindicated in bold in Tables 1 to 4.

Thus, in a preferred embodiment of the invention, on the basis of theamino acid residues present on the positions described in Tables 1 to 4,the amino acid sequence of a variable domain comprising the fullantigen-binding capacity in an antigen-binding protein of the inventioncan have the structure:

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 has an amino acid sequence chosen from the group        consisting of:

(SEQ ID: 197) a) [1]QVQLQESGGGLVQAGGSLRLSCAAS [25];b) an amino acid sequence that has at least 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100% sequence identity with the sequence in a); and/or,c) the amino acid sequence of a) that has one or more amino acidsubstitutions as defined in Table 1;in which FR2 is chosen from the group consisting of the amino acidsequence:

(SEQ ID: 198) d) [36]WFRQAPGKEREFVA [49];e) an amino acid sequence that has at least 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100% sequence identity with the sequence in d); and/orf) the amino acid sequence of d) that has one or more amino acidsubstitutions as defined in Table 2;in which FR3 is chosen from the group consisting of the amino acidsequence:

(SEQ ID: 199) g) [65]GRFTISRDNAKNTVYLQMDSLKPEDTAVYSCAA [94];h) an amino acid sequence that has at least 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100% sequence identity with the sequence in g); and/or,i) the amino acid sequence of g) that has one or more amino acidsubstitutions as defined in Table 3; and,in which FR4 is chosen from the group consisting of the amino acidsequence:

(SEQ ID: 200) j) [103]WGQGTQVTVSS [113];k) an amino acid sequence that has at least 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100% sequence identity with the sequence in j); and/or,l) the amino acid sequence of j) that has one or more amino acidsubstitutions as defined in Table 4.

In an alternative preferred embodiment, the antigen-binding protein ofthe invention comprises a CDR1, CDR2 and CDR3 combination as given inone of the rows of Table 5, wherein the framework regions (FR1 to FR4)may be any of the framework regions (FR1 to FR4) as defined above. Morepreferably, the antigen-binding protein of the invention comprises aCDR1, CDR2 and CDR3 combination as given in one of the rows of Table 5,wherein the antigen-binding protein has an amino acid sequence with atleast 90, 95, 98, 99 or 100% sequence identity to the sequence providedin the last cell of the corresponding row of Table 5.

The antigen-binding protein of the invention is a component thatspecifically binds to the target molecule with the desired bindingaffinity (as herein defined). The antigen-binding protein of theinvention preferably is a mono-specific antigen-binding protein. Acomposition comprising a mono-specific antigen-binding protein, such asthe immunoadsorbant materials of the present invention, is understood tomean a composition having a homogeneous population of theantigen-binding protein. It follows that the mono-specificantigen-binding protein is specific for a single epitope or ligand. Itis however expressly included in the invention that the immunoadsorbantmaterial may comprise more than one type of mono-specificantigen-binding protein, each consisting of a homogeneous population.Usually, however, in the context of the present invention, animmunoadsorbant material will not comprise more than 4, 6, 8, 10 or 20different mono-specific antigen-binding proteins. The antigen-bindingprotein will usually be an antibody or fragment thereof, in which casethe mono-specific antigen-binding protein will thus be a monoclonalantibody or a fragment thereof, which may be obtained from a clonedcell-line (e.g. hybridoma) or expressed from a cloned coding sequence.The term mono-specific antigen-binding protein as used herein thusexcludes polyclonal antibodies and antisera.

An antigen-binding protein of the invention, that can bind to, that hasaffinity for and/or that has specificity for a specific target molecule(antigenic determinant, epitope, antigen or protein) may be said to be“against” or “directed against” said target molecule. The term“specificity” refers to the number of different types of antigens orantigenic determinants to which a particular antigen-binding proteinmolecule can bind. The specificity of an antigen-binding protein can bedetermined based on affinity and/or avidity. The affinity, representedby the equilibrium constant for the dissociation of an antigen with anantigen-binding protein (K_(D)), is a measure for the binding strengthbetween an antigenic determinant and an antigen-binding site on theantigen-binding protein. Alternatively, the affinity can also beexpressed as the affinity constant (K_(A)), which is 1/K_(D). Affinitycan be determined in a manner known per se, depending on the specificcombination of antigen binding protein and antigen of interest. Avidityis herein understood to refer to the strength of binding of a targetmolecule with multiple binding sites by a larger complex of bindingagents, i.e. the strength of binding of multivalent binding. Avidity isrelated to both the affinity between an antigenic determinant and itsantigen binding site on the antigen-binding molecule and the number ofbinding sites present on the antigen-binding molecule. Affinity, on theother hand refers to simple monovalent receptor ligand systems.

Typically, antigen-binding proteins of the invention will bind thetarget molecule with a dissociation constant (K_(D)) of about 10⁻⁵ to10⁻¹² M or less, and preferably 10⁻⁷ to 10⁻¹² M or less and morepreferably 10⁻⁸ to 10⁻¹² M or less, and/or with a binding affinity of atleast 10⁻⁷ M, preferably at least 10⁻⁸ M, more preferably at least 10⁻⁹M, such as at least 10⁻¹⁰, 10⁻¹¹, 10⁻¹² M or more. Any K_(D) valuegreater than 10⁻⁴ M (i.e. less than 100 μM) is generally considered toindicate non-specific binding. Preferably, a polypeptide of theinvention will bind to the desired antigen with an affinity less than500 nM, preferably less than 200 nM, more preferably less than 10 nM,such as less than 500 pM. Specific binding of an antigen-binding proteinto an antigen or antigenic determinant can be determined in any suitablemanner known per se, including, for example, Scatchard analysis and/orcompetitive binding assays, such as radioimmunoassays (RIA), enzymeimmunoassays (EIA) and sandwich competition assays, and the differentvariants thereof known per se in the art. In a preferred embodiment theantigen-binding protein of the invention will bind to the desiredantigen with an affinity as defined above yet this affinity is combinedan efficient release of the antigen from the antigen-binding proteinunder mild elution conditions.

Mild elution conditions are herein understood to be conditions underwhich the activity and/or integrity (e.g. secondary/tertiary structure)are only slightly affected (e.g. less than 10% inactive or denatured),preferably there is no detectable reduction in activity and/or integrityof the antigen. Examples of such mild elution conditions include e.g.the acidic conditions as specified herein below, including e.g. 0.1 Mglycine pH 3.0 or pH 4.0, 0.1 M arginine pH 4.0 or pH 5.0. Otherexamples of mild elution conditions at (near)-neutral pH include e.g.high ionic strength such as condition equivalent 2 M NaCl (in e.g. 20 mMTris pH 8.0) or chaotropic agents such as ethylene glycol or propyleneglycol (40-60%, preferably about 50% (v/v), in e.g. 20 mM Imidazol, 10mM CaCl₂, 0.01% Tween 80, 250 mM NaCl at pH7.0). Examples ofantigen-binding proteins of the invention that release the antigen undermild elution condition as indicated above include antigen-bindingproteins that have a structure as herein defined above wherein: a) theCDR1 has an amino acid sequence selected from the group consisting ofSEQ ID No's: 1-9 and 16, and amino acid sequences that differs from SEQID No's: 1-9, and 16 in no more than 4, 3, 2, or 1 amino acid residues;b) the CDR2 has an amino acid sequence selected from the groupconsisting of SEQ ID No's: 50-58 and 65, and an amino acid sequencesthat differs from SEQ ID No's: 50-58 in no more than 6, 5, 4, 3, 2, or 1amino acid residues; and, c) the CDR3 has an amino acid sequenceselected from the group consisting of SEQ ID No's: 99-107 and an aminoacid sequences that differs from SEQ ID No's: 99-107 and 114, in no morethan 6, 5, 4, 3, 2, or 1 amino acid residues. More preferably theantigen-binding protein has an amino acid sequence selected from thegroup consisting of SEQ ID No's 148-156 and 163.

An epitope is defined as the portion of the target molecule that isbound by the antigen-binding protein. In case the antigen-bindingprotein is an antibody, the epitope is the portion of a target moleculethat triggers an immunological response upon immunisation of anindividual vertebrate host with this molecule. Generally it is the siteof the target molecule where binding to an antibody takes place. Theepitope is preferably present naturally in the target molecule.Optionally the epitope(s) is/are a sequence that has been artificiallyincluded in the target molecule. Optionally a multitude of the same ordifferent epitopes is included in the target molecule to facilitate itspurification and detection.

A target molecule is herein defined as a molecule that is to be bound bya binding agent, preferably an antigen-binding protein of the invention.A target molecule may be a protein that requires purification, or aprotein that is to be detected or identified. A preferred targetmolecule in the context of the present invention is a mammalian IgG.Preferably, the antigen-binding protein binds to the Fc (Fragmentcrystallizable) domain of a mammalian immunoglobulin. More preferablythe antigen-binding protein binds to Fc (Fragment crystallizable) domainof a mammalian IgG and does not bind to a mammalian immunoglobulin ofthe classes IgD, IgA, IgM or IgE. It is herein understood thatantigen-binding protein that binds to a first type of target moleculeand not to a second type of target molecule has a difference indissociation constants for the first and second types of targetmolecules, respectively of at least a factor 100, 1000, 10,000 or100,000. Preferably, the antigen-binding protein does not bind to theFab (Fragment antigen binding) domain of the mammalian immunoglobulin.Preferably, binding of the antigen-binding protein to the mammalianimmunoglobulin and subsequent elution of the immunoglobulin does notaffect effector functions of the mammalian immunoglobulin. Alsopreferred is that binding of the antigen-binding protein to themammalian immunoglobulin and subsequent elution of the immunoglobulindoes not reduce, inhibit or otherwise affect binding of the mammalianimmunoglobulin molecule to its predetermined antigen.

An antigen-binding protein of the invention that binds to an Fc domainof a mammalian IgG molecule is an antigen-binding protein thatpreferably has one or more properties selected from the group consistingof: a) the antigen-binding protein binds the human IgG molecule with abinding affinity of at least 10⁻⁷ M, 10⁻⁸ M, or 10⁻⁹ M as analyzed byBiaCore using polyclonal human IgG; b) the antigen-binding protein isobtainable by expression in yeast at an expression level of at least0.5, 0.8, 1.0 g/L of yeast culture; c) the antigen-binding protein has adynamic binding capacity of at least 2, 5 or 10 mg human IgG per ml ofcarrier, when coupled to NHS activated carrier (preferably Sepharose 4Bfast flow) at a density of 20 mg antigen-binding protein per ml NHScarrier and using a flow-rate of 150 cm/h; d) human IgG bound to theantigen-binding protein when coupled to reference NHS carrier as definedin c) is recovered from the antigen-binding protein with a yield of atleast 90, 95, or 99% using 0.1 M glycine, pH 2.0; e) human IgG bound tothe antigen-binding protein when coupled to reference NHS carrier asdefined in c) is recovered from the antigen-binding protein with a yieldof at least 70, 75, or 80% using 0.1 M glycine pH 3.0; f) theantigen-binding protein when coupled to NHS carrier as defined in c)retains a residual dynamic binding capacity of at least 70, 75 or 80%after 20 cleaning-in-place cycles, wherein in each cleaning-in-placecycle the antigen-binding protein coupled to reference NHS carrier iscontacted for 15 minutes with 0.05 M NaOH and 0.5 M NaCl at a flow rateof 150 cm/h; and g) the antigen-binding protein can be immobilized ontocarriers and/or carriers via standard coupling chemistries (e.g. NHS orCNBr activated carriers) and still retain the functionality of IgGbinding (i.e. has a dynamic binding capacity as defined in c)) withoutthe need of additional tags or linkers genetically incorporated at theN- and/or C-terminus of the antigen-binding protein. More preferably,the antigen-binding protein has at least 4, 5, 6, or all of saidproperties. For convenience, throughout this specification a referenceto “reference NHS carrier” refers to the NHS activated carrier(preferably Sepharose 4B fast flow) at a density of 20 mgantigen-binding protein per ml NHS carrier as defined in c) above.

A preferred antigen-binding protein of the invention binds to the Fcdomain of a human IgG molecule but does not bind to an IgG molecule ofmurine or bovine origin. A preferred antigen-binding protein binds toone or more of human IgG1, IgG2, IgG3 and IgG4 molecules, morepreferably the antigen-binding protein binds to all four human IgGsubclasses. An antigen-binding protein that binds to the Fc domain of ahuman IgG molecule but does not bind to an IgG molecule of murine orbovine origin preferably is an antigen-binding protein having astructure as herein defined above wherein: a) the CDR1 has an amino acidsequence selected from the group consisting of SEQ ID No's: 1-15, 17-25,31-36, 38, 43 and 44 and amino acid sequences that differs from SEQ IDNo's: 1-15, 17-25, 31-36, 38, 43 and 44 in no more than 4, 3, 2 or 1amino acid residues; b) the CDR2 has an amino acid sequence selectedfrom the group consisting of SEQ ID No's: 50-64, 66-74, 80-85, 87, 92and 93 and an amino acid sequences that differs from SEQ ID No's: 50-64,66-74, 80-85, 87, 92 and 93 in no more than 6, 5, 4, 3, 2, or 1 aminoacid residues; and, c) the CDR3 has an amino acid sequence selected fromthe group consisting of SEQ ID No's: 99-113, 115-123, 129-134, 136, 141and 142 and an amino acid sequences that differs from SEQ ID No's:99-113, 115-123, 129-134, 136, 141 and 142 in no more than 6, 5, 4, 3,2, or 1 amino acid residues. More preferably the antigen-binding proteinhas an amino acid sequence selected from the group consisting of SEQ IDNo's 148-162, 164-172, 178-183, 185, 190 and 191.

A more preferred antigen-binding protein of the invention binds to theFc domain of a human IgG molecule but does not bind to an IgG moleculeof murine, bovine or caprine (goat) origin. The antigen-binding proteinpreferably binds to one or more of human IgG1, IgG2, IgG3 and IgG4molecules, more preferably the antigen-binding protein binds to all fourhuman IgG subclasses. An antigen-binding protein that binds to the Fcdomain of a human IgG molecule but does not bind to an IgG molecule ofmurine, bovine or caprine origin preferably is an antigen-bindingprotein having a structure as herein defined above wherein: a) the CDR1has an amino acid sequence selected from the group consisting of SEQ IDNo's: 1-15, 17-25, 31-36, 38 and 44 and amino acid sequences thatdiffers from SEQ ID No's: 1-15, 17-25, 31-36, 38 and 44 in no more than4, 3, 2 or 1 amino acid residues; b) the CDR2 has an amino acid sequenceselected from the group consisting of SEQ ID No's: 50-64, 66-74, 80-85,87 and 93 and an amino acid sequences that differs from SEQ ID No's:50-64, 66-74, 80-85, 87 and 93 in no more than 6, 5, 4, 3, 2, or 1 aminoacid residues; and, c) the CDR3 has an amino acid sequence selected fromthe group consisting of SEQ ID No's: 99-113, 115-123, 129-134, 136 and142 and an amino acid sequences that differs from SEQ ID No's: 99-113,115-123, 129-134, 136 and 142 in no more than 6, 5, 4, 3, 2, or 1 aminoacid residues. More preferably the antigen-binding protein has an aminoacid sequence selected from the group consisting of SEQ ID No's 148-162,164-172, 178-183, 185 and 191.

An even more preferred antigen-binding protein of the invention binds tothe Fc domain of a human IgG molecule but does not bind to an IgGmolecule that originates from murine, bovine, caprine, rat, syrianhamster, guinea pig, dog, cat or sheep. The antigen-binding proteinpreferably binds to one or more of human IgG1, IgG2, IgG3 and IgG4molecules, more preferably the antigen-binding protein binds to all fourhuman IgG subclasses. The antigen-binding protein further preferably hasone or more properties selected from the group consisting of: a) theantigen-binding protein binds the human IgG molecule with a bindingaffinity of at least 5 nM as analyzed by BiaCore using polyclonal humanIgG; b) human IgG bound to the antigen-binding protein when coupled toreference NHS carrier is recovered from the antigen-binding protein witha yield of at least 99% using 0.1 M glycine, pH 2.0; and, c) human IgGbound to the antigen-binding protein when coupled to reference NHScarrier is recovered from the antigen-binding protein with a yield of atleast 80% using 0.1 M glycine pH 3.0. More preferably theantigen-binding protein preferably has at least 2 or 3 of saidproperties. Such an antigen-binding protein further preferably has astructure as herein defined above wherein: a) the CDR1 has an amino acidsequence selected from the group consisting of SEQ ID No's: 1-15 andamino acid sequences that differs from SEQ ID No's: 1-15 in no more than4, 3, 2 or 1 amino acid residues; b) the CDR2 has an amino acid sequenceselected from the group consisting of SEQ ID No's: 50-64 and an aminoacid sequences that differs from SEQ ID No's: 50-64 in no more than 6,5, 4, 3, 2, or 1 amino acid residues; and, c) the CDR3 has an amino acidsequence selected from the group consisting of SEQ ID No's: 99-113 andan amino acid sequences that differs from SEQ ID No's: 99-113 in no morethan 6, 5, 4, 3, 2, or 1 amino acid residues. More preferably theantigen-binding protein has an amino acid sequence selected from thegroup consisting of SEQ ID No's 148-162.

An most preferred antigen-binding protein of the invention binds to theFc domain of a human IgG molecule but does not bind to an IgG moleculethat originates from murine, bovine, caprine, rat, syrian hamster,guinea pig, dog, cat or sheep. The antigen-binding protein preferablybinds to one or more of human IgG1, IgG2, IgG3 and IgG4 molecules, morepreferably the antigen-binding protein binds to all four human IgGsubclasses. The antigen-binding protein further preferably has one ormore properties selected from the group consisting of: a) theantigen-binding protein binds the human IgG molecule with a bindingaffinity of at least 5 nM as analyzed by BiaCore using polyclonal humanIgG; b) human IgG bound to the antigen-binding protein when coupled toreference NHS carrier is recovered from the antigen-binding protein witha yield of at least 99% using 0.1 M glycine, pH 3.0; c) human IgG boundto the antigen-binding protein when coupled to reference NHS carrier isrecovered from the antigen-binding protein with a yield of at least 95%using 0.1 M glycine, pH 4.0; d) human IgG bound to the antigen-bindingprotein when coupled to reference NHS carrier is recovered from theantigen-binding protein with a yield of at least 99% using 0.1-0.2 Marginine, pH 3.0; e) retains a residual dynamic binding capacity of atleast 90, 95 or 100% after 100 cleaning-in-place cycles, wherein in eachcleaning-in-place cycle the antigen-binding protein coupled to referenceNHS carrier is contacted for 15 minutes with 0.1 M NaOH at a flow rateof 150 cm/h; and f) retains a residual dynamic binding capacity of atleast 80% after 40 cleaning-in-place cycles, wherein in eachcleaning-in-place cycle the antigen-binding protein coupled to referenceNHS carrier is contacted for 15 minutes with 0.2 M NaOH at a flow rateof 150 cm/h. More preferably the antigen-binding protein preferably hasat least 2, 3, 4, 5 or 6 of said properties. Such an antigen-bindingprotein further preferably has a structure as herein defined abovewherein: a) the CDR1 has an amino acid sequence selected from the groupconsisting of SEQ ID No's: 1-9 and amino acid sequences that differsfrom SEQ ID No's: 1-9 in no more than 4, 3, 2, or 1 amino acid residues;b) the CDR2 has an amino acid sequence selected from the groupconsisting of SEQ ID No's: 50-58 and an amino acid sequences thatdiffers from SEQ ID No's: 50-58 in no more than 6, 5, 4, 3, 2, or 1amino acid residues; and, c) the CDR3 has an amino acid sequenceselected from the group consisting of SEQ ID No's: 99-107 and an aminoacid sequences that differs from SEQ ID No's: 99-107 in no more than 6,5, 4, 3, 2, or 1 amino acid residues. More preferably theantigen-binding protein has an amino acid sequence selected from thegroup consisting of SEQ ID No's 148-156.

An alternatively more preferred antigen-binding protein of the inventionbinds to the Fc domain of a human IgG molecule but does not bind to anIgG molecule that originates from murine, bovine, caprine, rat, syrianhamster, guinea pig, dog, cat or sheep. The antigen-binding proteinpreferably binds to one or more of human IgG1, IgG2, IgG3 and IgG4molecules, more preferably the antigen-binding protein binds to all fourhuman IgG subclasses. The antigen-binding protein further preferably hasone or more properties selected from the group consisting of: a) theantigen-binding protein binds the human IgG molecule with a bindingaffinity of at least 3 nM as analyzed by BiaCore using polyclonal humanIgG; and b) the antigen-binding protein is obtainable by expression inyeast at an expression level of at least 1.2 g/L of yeast culture. Morepreferably the antigen-binding protein preferably has both of saidproperties. Such an antigen-binding protein further preferably has astructure as herein defined above wherein: a) the CDR1 has an amino acidsequence selected from the group consisting of SEQ ID No's: 10-15 andamino acid sequences that differs from SEQ ID No's: 10-15 in no morethan 4, 3, 2, or 1 amino acid residues; b) the CDR2 has an amino acidsequence selected from the group consisting of SEQ ID No's: 59-64 and anamino acid sequences that differs from SEQ ID No's: 59-64 in no morethan 6, 5, 4, 3, 2, or 1 amino acid residues; and, c) the CDR3 has anamino acid sequence selected from the group consisting of SEQ ID No's:108-113 and an amino acid sequences that differs from SEQ ID No's:108-113 in no more than 6, 5, 4, 3, 2, or 1 amino acid residues. Morepreferably the antigen-binding protein has an amino acid sequenceselected from the group consisting of SEQ ID No's 157-162.

A preferred alternative antigen-binding protein of the invention bindsto the Fc domain of an IgG molecule from at least two different speciesselected from the group consisting of human, murine, and bovine. Such apreferred alternative antigen-binding protein preferably has a structureas herein defined above wherein: a) the CDR1 has an amino acid sequenceselected from the group consisting of SEQ ID No's: 16, 26-30, 37, 42 and47-49 and amino acid sequences that differs from SEQ ID No's: 16, 26-30,37, 42 and 47-49 in no more than 4, 3, 2 or 1 amino acid residues; b)the CDR2 has an amino acid sequence selected from the group consistingof SEQ ID No's: 65, 75-79, 86, 91 and 96-98 and an amino acid sequencesthat differs from SEQ ID No's: 65, 75-79, 86, 91 and 96-98 in no morethan 6, 5, 4, 3, 2, or 1 amino acid residues; and, c) the CDR3 has anamino acid sequence selected from the group consisting of SEQ ID No's:114, 124-128, 135, 140 and 145-147 and an amino acid sequences thatdiffers from SEQ ID No's: 114, 124-128, 135, 140 and 145-147 in no morethan 6, 5, 4, 3, 2, or 1 amino acid residues. More preferably theantigen-binding protein has an amino acid sequence selected from thegroup consisting of SEQ ID No's 163, 173-177, 184, 189 and 194-196.

A more preferred alternative antigen-binding protein of the inventionbinds to the Fc domain of an IgG molecule from a human, murine, bovine,rat, rabbit, dog, cat, swine, sheep, primate (of which at leastchimpanzee and rhesus), donkey, and horse. Such a preferred alternativeantigen-binding protein preferably has a structure as herein definedabove wherein: a) the CDR1 has an amino acid sequence selected from thegroup consisting of SEQ ID No's: 16, 28-30, 37, 42 and 47-49 and aminoacid sequences that differs from SEQ ID No's: 16, 28-30, 37, 42 and47-49 in no more than 4, 3, 2 or 1 amino acid residues; b) the CDR2 hasan amino acid sequence selected from the group consisting of SEQ IDNo's: 65, 77-79, 86, 91 and 96-98 and an amino acid sequences thatdiffers from SEQ ID No's: 65, 77-79, 86, 91 and 96-98 in no more than 6,5, 4, 3, 2, or 1 amino acid residues; and, c) the CDR3 has an amino acidsequence selected from the group consisting of SEQ ID No's: 114,126-128, 135, 140 and 145-147 and an amino acid sequences that differsfrom SEQ ID No's: 114, 126-128, 135, 140 and 145-147 in no more than 6,5, 4, 3, 2, or 1 amino acid residues. More preferably theantigen-binding protein has an amino acid sequence selected from thegroup consisting of SEQ ID No's 163, 175-177, 184, 189 and 194-196.

The most preferred alternative antigen-binding protein of the inventionis a “multi-species antigen-binding protein” that binds to the Fc domainof an IgG molecule from human, bovine, mouse, rat, rabbit, dog, cat,swine, sheep, primate (chimpanzee, rhesus), donkey, horse, goat, syrianhamster, guinea pig. More preferably this antigen-binding protein bindsto the Fc domain of an IgG molecule from at least some, or preferablyall species within the orders carnivores, even- and odd-toed ungulates,primates, rodents and Lagomorpha (including rabbits), most preferablythis antigen-binding protein binds to the Fc domain of an IgG moleculefrom all mammalian species. The antigen-binding protein furtherpreferably has one or more properties selected from the group consistingof: a) the antigen-binding protein binds the human IgG molecule with abinding affinity of at least 20 nM as analyzed by BiaCore usingpolyclonal human IgG; b) the antigen-binding protein is obtainable byexpression in yeast at an expression level of at least 2.5 g/L of yeastculture; and c) human IgG bound to the antigen-binding protein whencoupled to reference NHS carrier is recovered from the antigen-bindingprotein with a yield of at least 99% using 0.1 M glycine, pH 3.0 or 0.2M arginine, pH 3.0. More preferably the antigen-binding proteinpreferably has at least 2 or 3 of said properties. The most preferredalternative antigen-binding protein preferably has a structure as hereindefined above wherein a) the CDR1 has an amino acid sequence selectedfrom the group consisting of SEQ ID No's: 16, 28-30 and 48 or an aminoacid sequences that differs from SEQ ID No's: 16, 28-30 and 48 in nomore than 4, 3, 2, or 1 amino acid residues; b) the CDR2 has an aminoacid sequence selected from the group consisting of SEQ ID No's: 65,77-79 and 97 or an amino acid sequences that differs from SEQ ID No's:65, 77-79 and 97 in no more than 6, 5, 4, 3, 2, or 1 amino acidresidues; and, c) the CDR3 has an amino acid sequence selected from thegroup consisting of SEQ ID No's: 114, 126-128 and 146 or an amino acidsequences that differs from SEQ ID No's: 114, 126-128 and 146 in no morethan 6, 5, 4, 3, 2, or 1 amino acid residues. More preferably theantigen-binding protein has an amino acid sequence selected from thegroup consisting of SEQ ID No: 163, 175-177 and 195.

In one embodiment the invention pertains to particular form of anantigen-binding protein of the invention: a multivalent antigen-bindingprotein. The multivalent antigen-binding protein comprises the aminoacid sequences of at least two antigen-binding proteins as definedherein above. The amino acid sequences of at least two antigen-bindingproteins may be different from each or they may be identical, e.g.copies or repeats of one amino acid sequence. The amino acid sequencesof the at least two antigen-binding proteins will usually be fusedhead-to tail, i.e. the C-terminus of the most N-terminal sequence fusedto the N-terminus of the second sequence and so on. The amino acidsequences of at least two antigen-binding proteins may be fused directlylinked or via a linker or spacer. Multivalent antigen-binding proteinsof the invention may be produced by expression of a nucleotide sequenceencoding the multivalent protein wherein two or more coding sequences ofthe antigen-binding proteins are operably linked together in the samereading frame. The skilled person will know how to operably fuse proteincoding sequences.

In a further aspect the invention relates to a fusion protein whereinthe amino acid sequence of an antigen-binding proteins as defined hereinis fused with an amino acid sequence of a therapeutic protein. The twoamino acid sequences are preferably linked together by a genetic fusionwherein nucleotide sequences encoding the respective amino acidsequences are operably linked together in frame by means known per se inthe art. The amino acid sequences may be linked directly or optionallythrough a spacer or linker amino acid sequence. The fusion proteinscomprising an amino acid sequence of an antigen-binding protein of theinvention fused to a therapeutic protein are useful in increasing theserum half-life of the proteins. Injected biotherapeutics may be rapidlycleared from the blood circulation after administration, requiring highdoses or frequent administration to maintain effective therapeuticlevels. To overcome these problems, the biotherapeutic proteins orpeptides can be bound to circulating serum proteins such as IgGs toenhance their bioavailability. In the present invention thebiotherapeutic proteins or peptides are bound to circulating IgGs byfusing the amino acid sequence of the biotherapeutic protein or peptideto that of an antigen-binding protein of the invention. This willenhance the bioavailability of the fused biotherapeutic protein orpeptide. The genetic fusion of the antigen-binding proteins of theinvention to biotherapeutics can provide a binding moiety directed tothe Fc domain of IgG, resulting in increased half-life of thebiotherapeutic in serum. Harmsen et al., (2005, Vaccine 23 (41), p.4926-42) have indeed reported that binding of a model VHH withtherapeutic potential to porcine IgG through a fusion with a VHH thatbinds porcine IgG resulted in an increase in the in vivo residence ofthe model VHH compared to a control fusion VHH that did not bind toporcine IgG. This method of improving serum half-life may be applied inprinciple to any biotherapeutic protein, including e.g. antigens (forvaccination), enzymes (for enzyme replacement therapy), hormones,chymokines, interleukins, (humanised) monoclonal antibodies, and thelike.

In another aspect the invention relates to a nucleic acid comprising anucleotide sequence encoding an antigen-binding protein as definedherein above. A preferred nucleic acid according to the invention is anucleic acid construct, wherein the nucleotide sequence encoding theantigen-binding protein is operably linked to a promoter and optionallyother regulatory elements such as e.g. terminators, enhancers,polyadenylation signals, signal sequences for secretion and the like.Such nucleic acid constructs are particularly useful for the productionof the antigen-binding proteins of the invention using recombinanttechniques in which a nucleotide sequence encoding the antigen-bindingprotein of interest is expressed in suitable host cells such asdescribed in Ausubel et al., “Current Protocols in Molecular Biology”,Greene Publishing and Wiley-Interscience, New York (1987) and inSambrook and Russell (2001) “Molecular Cloning: A Laboratory Manual(3^(rd) edition), Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, New York). As used herein, the term “operably linked”refers to a linkage of polynucleotide elements in a functionalrelationship. A nucleic acid is “operably linked” when it is placed intoa functional relationship with another nucleic acid sequence. Forinstance, a promoter or enhancer is operably linked to a coding sequenceif it affects the transcription of the coding sequence. Operably linkedmeans that the DNA sequences being linked are typically contiguous and,where necessary to join two protein coding regions, contiguous and inreading frame.

In a further aspect the invention pertains to a host cell comprising anucleic acid as defined above. Preferably the host cell is a host cellfor production of antigen-binding protein of the invention. The hostcell may be any host cell capable of producing an antigen-bindingprotein of the invention, including e.g. a prokaryotic host cell, suchas e.g., E. coli, or a (cultured) mammalian, plant, insect, fungal oryeast host cell, including e.g. CHO-cells, BHK-cells, human cell lines(including HeLa, COS and PER.C6), Sf9 cells and Sf+ cells. A preferredhost cell for production of an antigen-binding protein of the inventionis however a cell of an eukaryotic microorganism such as yeasts andfilamentous fungi. Preferred yeast host cell e.g. include e.g.Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, andKluyveromyces lactis. Preferred strains, constructs and fermentationconditions for production of the antigen-binding protein of theinvention are described by van de Laar, et al., (2007, Biotechnology andBioengineering, Vol. 96, No. 3: 483-494). For example, production of theantigen-binding proteins can be performed in standard bioreactors with aworking volume between 10 and 10,000 litres. Dissolved oxygen can becontrolled by automatic adjustment of the impeller speed. The pH can becontrolled using phosphoric acid and ammoniac gas or ammonia solutionand temperature controlled via e.g. a cooling jacket and heating jacket.The offgas is analysed on ethanol concentration, rO₂ and rCO₂. The batchphase is started by adding 3%-8% of full-grown inoculum (e.g. 30° C.,0.3-0.4 VVM air, DO₂ minimum 30%, pH 5.0). When the ethanolconcentration in offgas is declining in batch phase the ethanolfermentation can be started. The feed can be applied according to apulsed feed profile to maintain the ethanol level within the demandedmargins. The feed phases can be performed at 21° C. and 0.7-1.1 VVM air.During the ethanol fermentations DO₂ decreases to 0% and accumulatedethanol can be further controlled by a pulsed feed profile. Feed phasestops when the ethanol feed is depleted. The broth can be chilled to atemperature between 5-10° C. till further processing like biomassremoval etc. (VVM=volumes of air per minute per volume of batch). Inthis context it is also understood that whenever herein we anantigen-binding protein of the invention as being obtainable byexpression in yeast at a certain minimal expression level, this level isobtained using the method as described in Example 1.1. herein, wherebythe (maximal) concentration of the antigen-binding protein (at the endof fermentation) “g/L” refers to the amount of secreted antigen-bindingprotein (in grams) per litre of cell-free broth (i.e., after removal ofbiomass by e.g. filtration).

Examples of antigen-binding proteins of the invention that areobtainable by expression in yeast at an expression level of at least 1.2g/L of yeast culture include antigen-binding proteins that have astructure as herein defined above wherein: a) the CDR1 has an amino acidsequence selected from the group consisting of SEQ ID No's: 10-16 andamino acid sequences that differs from SEQ ID No's: 10-16 in no morethan 4, 3, 2, or 1 amino acid residues; b) the CDR2 has an amino acidsequence selected from the group consisting of SEQ ID No's: 59-65 and anamino acid sequences that differs from SEQ ID No's: 59-65 in no morethan 6, 5, 4, 3, 2, or 1 amino acid residues; and, c) the CDR3 has anamino acid sequence selected from the group consisting of SEQ ID No's:108-114 and an amino acid sequences that differs from SEQ ID No's:108-114 in no more than 6, 5, 4, 3, 2, or 1 amino acid residues. Morepreferably the antigen-binding protein has an amino acid sequenceselected from the group consisting of SEQ ID No's 157-163.

Thus, in yet another aspect the invention relates to a method forproducing an antigen-binding protein of the invention, wherein themethod preferably comprises the steps of: a) culturing a host cell asdefined above under conditions conducive to expression of theantigen-binding protein; and optionally, b) purifying theantigen-binding protein from at least one of the host cell and theculture medium. Suitable conditions may include the use of a suitablemedium, the presence of a suitable source of food and/or suitablenutrients, a suitable temperature, and optionally the presence of asuitable inducing factor or compound (e.g. when the nucleotide sequencesof the invention are under the control of an inducible promoter); all ofwhich may be selected by the skilled person. Under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced. Theantigen-binding proteins of the invention may then be isolated from thehost cell/host organism and/or from the medium in which said host cellor host organism was cultivated, using protein isolation and/orpurification techniques known per se, such as (preparative)chromatography and/or electrophoresis techniques, differentialprecipitation techniques, affinity techniques (e.g. using a specific,cleavable amino acid sequence fused with the amino acid sequence of theinvention) and/or preparative immunological techniques (i.e. usingantibodies against the antigen-binding protein to be isolated).

In one aspect the invention also relates to a composition comprising anantigen-binding protein as defined herein. A preferred embodimentthereof is an immunoadsorbent material comprising the antigen-bindingprotein. An immunoadsorbent material is herein understood to mean thecombination of a carrier and an antigen-binding protein that isimmobilized on the carrier. Preferably in the immunoadsorbent materialthe antigen-binding protein is immobilized onto a carrier, whereby morepreferably, the antigen-binding protein is immobilised onto the carrierby a covalent link. The carrier may be any material that may be used tofor immobilization of an antigen-binding protein. Suitable examples arematrix materials, to entrap the binding agent, cell surfaces on whichthe binding agent is displayed and polymers that can be covalentlylinked to the binding agent. The person skilled in the art of affinitychromatography is well aware of suitable carriers such as e.g. poroussolid phase carrier materials such as agarose, polystyrene, controlledpore glass, cellulose, dextrans, kieselguhr, synthetic polymers such asSepharose™, porous amorphous silica. The carrier materials may be in anysuitable format such as particles, powders, sheets, beads, filters andthe like. Further specifications of suitable carrier materials are forexample disclosed in EP-A-434317. Methods are available for immobilizingligands quickly, easily and safely through a chosen functional group.The correct choice of coupling method depends on the substance toimmobilized. For example the following commercially known derivatives ofSepharose™ allow the convenient immobilization of proteins thereon:CNBr-activated Sepharose™ 4B enables ligands containing primary aminogroups to be rapidly immobilized by a spontaneous reaction.AH-Sepharose™ 4B and CH-Sepharose™ 4B both have a six-carbon long spacerarm and permit coupling via carboxyl and amino groups respectively.Flexible spacers are suitable for use in situations where theflexibility of the target molecules is limited or where 3-dimensionalstructure of the target requires some flexibility of the binding agentto allow optimal binding. Activated CH-Sepharose™ 4B provides asix-carbon spacer arm and an active ester for spontaneous coupling viaamino groups. These are only a few examples of suitable immobilisationroutes. Optionally the immunoadsorbent material is put into a column tofacilitate easy chromatographic separations.

In yet a further aspect the invention relates to a method for thepurification of a mammalian IgG molecule. The method preferablycomprises the steps of: a) bringing a composition comprising a targetmolecule, e.g. a mammalian IgG molecule, in contact with theimmunoadsorbent material comprising an antigen-binding protein of theinvention, preferably under conditions that allow binding of the targetmolecule to the immunoadsorbent material; b) optionally, performing oneor more washing steps; c) eluting the bound target molecule underconditions that decrease the affinity between the target molecule andthe immunoadsorbent material; and, d) optionally, further processingtarget molecule.

The composition comprising the target molecule will often be an aqueouscomposition comprising many other proteins besides the target that is tobe purified. The conditions of the contact step are preferably such thatbinding of the binding agent, to the target molecule occurs. Preferablyin this step a loading buffer having pH around 6.5 to 8 is used. Asuitable buffer is e.g. a PBS buffer or similar buffer a physiologicalionic strength and pH. It is preferred that the loaded material washeduntil the non specific binders have eluted. This is usually done byrinsing with a suitable buffer, which may be the same as the loadingbuffer. Desorption or elution of the target molecule is the next step.This is preferably done by changing the conditions such that theantibody or fragment no longer binds the target molecule. Elution may beachieved by changing the conditions with respect to pH, salt,temperature or any other suitable measure. A preferred elution methodfor desorption is elution with a buffer having a pH below 4, 3 or 2.Suitable elution buffers are described herein above.

More specifically the invention relates to a method for the purificationof a target molecule by immunoaffinity comprising the steps of: a)selecting an antigen-binding protein or fragment thereof, that binds tothe target molecule; b) binding the antigen-binding protein or fragmentthereof to immunonoadsorbent material; c) loading the immunoadsorbentmaterial with a composition comprising the target molecule, preferablyunder conditions where binding of the antigen-binding protein to thetarget molecule takes place; d) washing the loaded immunoadsorbent toremove non specific binders; and, e) eluting the target molecule byapplying elution conditions. Preferably a fragment of theantigen-binding protein retains binding affinity as defined above in thecontext of this invention.

In again a further aspect, the invention pertain to the use of anantigen-binding protein as defined herein for the detection and/orpurification of a mammalian IgG molecule.

In one aspect the invention relates to methods for therapeuticapheresis. Therapeutic apheresis is an extracorporeal blood treatment toeliminate pathogenic compounds from the blood (Bosch, 2003, J. Artif.Organs 6(1): 1-8). One example of TA concerns the adsorption ofantibodies in a variety of antibody-mediated immune diseases. A commonlyused matrix for adsorption of antibodies in TA is Protein A sepharose.This matrix is used for the treatment of various auto-immune diseasesand antibody-mediated transplant rejections. However, due to lowaffinity for human IgG subclass 3 antibodies, Protein A matrix is notefficient in the removal of IgG3 antibodies. Advantageously, theantigen-binding proteins of the present invention that are specific formammalian or human IgG can also be used for the depletion of IgG,including IgG3, in patients suffering from antibody-mediated diseases.Preferably the method for therapeutic apheresis comprises at least oneof removing, depleting and inactivating mammalian IgG in (from) a bodyfluid. Preferably the removing, depleting and inactivating of mammalianIgG in (from) a body fluid is performed ex vivo. The body fluidpreferably is blood, a blood fraction such as e.g. blood plasma or bloodserum, or another body fluid. In the method an antigen-binding proteinof the invention as defined hereinabove or an immunoadsorbent materialcomprising the antigen-binding protein as defined above, is brought intoextracorporeal contact with the body fluid of a subject, preferably ahuman subject. The immunoadsorbent apheresis material may be in the formof particles or beads, which may advantageously be packed into a flowchamber or a column, through which the body fluid of the subject orpatient is passed extracorporeally. Before or after a treatment in whichIgG is depleted, one or more further treatment stages for the body fluidcan be carried out. Several treatments of the body fluid can be carriedout in successive units, in which IgG is depleted by adsorption, toachieve the desired end concentration of IgG. Samples of the body fluidbefore and after IgG depletion may be tested using e.g. ELISA for IgGlevels (using e.g. the antigen-binding proteins of the invention). Thebody fluid may then be reinfused into the subject or human patient,although the latter step may be explicitly excluded from a preferredextracorporal embodiment of the method. In preferred embodiments themethods of the invention for therapeutic apheresis are applied on bodyfluids from patient or subjects suffering from an antibody-mediatedautoimmune disease, antibody-mediated transplant rejection or anautoimmune disease with an antibody-mediated component. Examples of suchdiseases include Myasthenia gravis, Goodpasture syndrome, Systemic LupusErythematosis (SLE) and dilated cardiomyopathy (DCM). The aphereticmethods of the invention are particularly useful for autoimmune diseasesin which auto-antibodies of subclass 3 are involved, like e.g. SLE andDCM, as IgG3 is not efficiently depleted using Protein A (Staudt et al.,2002, Circulation 106: 2448-2453).

In one aspect the invention thus also pertains to the use of anantigen-binding protein of the invention that binds a mammalian IgGmolecule for extracorporeal removal or depletion of mammalian IgG in asubject's body fluid, preferably a human subject.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

TABLE 1 Non-limiting examples of amino acid residues in FR1 Position 1 23 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25a.a.residue Q V Q L Q E S G G G L V Q A G G S L R L S C A A S anti-IgGFc D P D V K V A V P L a.a. sequences T E R V S D a.a.residue A K E Q FD M A E Q A F L F F D I A Camelid VHH's E L A R S K A N I T E L F V V PG S P S P W R S T T T T V

TABLE 2 Non-limiting examples of amino acid residues in FR2 Position 3637 38 39 40 41 42 43 44 45 46 47 48 49 a.a.residue W F R Q A P G K E R EF V A anti-IgG Fc a.a. sequences Y R L L N Q L L L S H E T T G S G I P AA V V G G W a.a.residue L H F A E D A C D I I T Camelid VHH's P S E D IK M V Q R L Q R R S P V V V L Q Y V

TABLE #3 Non-limiting examples of amino acid residues in FR3 Position 6566 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 a.a.residue G R F T I SR D N A K N T V Y L Q anti-IgG Fc A A V F G E R T Q D M M F E a.a.sequences D T Y K S V G Y A I H V N K P N T A D L M Y G H K G S S G E EN L F a.a.residue L N L A H G A D A R E F A F I Camelid VHH's V S M T IN D N L S I V R L V F S R P T Q I S T L W T V Position 82 82a 82b 82c 8384 85 86 87 88 89 90 91 92 93 94 a.a.residue M N S L K P E D T A V Y Y CA A anti-IgG Fc L S N I Q A D G M F N I a.a. sequences I T E L I L H G VD R V S S V R F R Y T S F a.a.residue G D P N D G A S A F D G V CamelidH G V G R Q S D H K C VHH's R I S L T R F T M T M V T K T N L T S

TABLE 4 Non-limiting examples of amino acid residues in FR4 Position 103104 105 106 107 108 109 110 111 112 113 a.a.residue W G Q G T Q V T V SS anti-IgG Fc a.a. sequences R K L I A A P N a.a.residue P D E A R I A FA Camelid VHH's R R I I L S P T

EXAMPLES Example 1 Identification of IgG-Fc Domain Binding VHH Fragments

The IgG-Fc domain binding VHH fragments were identified from llamasimmunized with mammalian IgG antibodies and/or Fc fragments thereof.Screening of individual VHH fragments was performed by ELISA using IgGfrom different mammalian species and/or Fc- and Fab fragments thereof,including non-IgG antibodies like IgM and IgA, which resulted in a panelof VHH fragments binding to the Fc domain of mammalian IgG and human IgGin particular. Table 5 presents the CDR1, CDR2 and CDR3 amino acidsequences that are comprised in each of the VHH fragments and also theamino acid sequence of each of the VHH fragments including the frameworkregions.

TABLE 5 CDR1, CDR2 and CDR3 amino acid sequences in each of the clonesand the amino acid sequence of each clone including the frameworkregions (FR) VHH CDR1 CDR2 CDR3 VHH fragment including FR IgG-Fc-01 SEQID NO: 1 SEQ ID NO: 50 SEQ ID NO: 99 SEQ ID NO: 148 IgG-Fc-02 SEQ ID NO:2 SEQ ID NO: 51 SEQ ID NO: 100 SEQ ID NO: 149 IgG-Fc-03 SEQ ID NO: 3 SEQID NO: 52 SEQ ID NO: 101 SEQ ID NO: 150 IgG-Fc-04 SEQ ID NO: 4 SEQ IDNO: 53 SEQ ID NO: 102 SEQ ID NO: 151 IgG-Fc-05 SEQ ID NO: 5 SEQ ID NO:54 SEQ ID NO: 103 SEQ ID NO: 152 IgG-Fc-06 SEQ ID NO: 6 SEQ ID NO: 55SEQ ID NO: 104 SEQ ID NO: 153 IgG-Fc-07 SEQ ID NO: 7 SEQ ID NO: 56 SEQID NO: 105 SEQ ID NO: 154 IgG-Fc-08 SEQ ID NO: 8 SEQ ID NO: 57 SEQ IDNO: 106 SEQ ID NO: 155 IgG-Fc-09 SEQ ID NO: 9 SEQ ID NO: 58 SEQ ID NO:107 SEQ ID NO: 156 IgG-Fc-10 SEQ ID NO: 10 SEQ ID NO: 59 SEQ ID NO: 108SEQ ID NO: 157 IgG-Fc-11 SEQ ID NO: 11 SEQ ID NO: 60 SEQ ID NO: 109 SEQID NO: 158 IgG-Fc-12 SEQ ID NO: 12 SEQ ID NO: 61 SEQ ID NO: 110 SEQ IDNO: 159 IgG-Fc-13 SEQ ID NO: 13 SEQ ID NO: 62 SEQ ID NO: 111 SEQ ID NO:160 IgG-Fc-14 SEQ ID NO: 14 SEQ ID NO: 63 SEQ ID NO: 112 SEQ ID NO: 161IgG-Fc-15 SEQ ID NO: 15 SEQ ID NO: 64 SEQ ID NO: 113 SEQ ID NO: 162IgG-Fc-16 SEQ ID NO: 16 SEQ ID NO: 65 SEQ ID NO: 114 SEQ ID NO: 163IgG-Fc-17 SEQ ID NO: 17 SEQ ID NO: 66 SEQ ID NO: 115 SEQ ID NO: 164IgG-Fc-18 SEQ ID NO: 18 SEQ ID NO: 67 SEQ ID NO: 116 SEQ ID NO: 165IgG-Fc-19 SEQ ID NO: 19 SEQ ID NO: 68 SEQ ID NO: 117 SEQ ID NO: 166IgG-Fc-20 SEQ ID NO: 20 SEQ ID NO: 69 SEQ ID NO: 118 SEQ ID NO: 167IgG-Fc-21 SEQ ID NO: 21 SEQ ID NO: 70 SEQ ID NO: 119 SEQ ID NO: 168IgG-Fc-22 SEQ ID NO: 22 SEQ ID NO: 71 SEQ ID NO: 120 SEQ ID NO: 169IgG-Fc-23 SEQ ID NO: 23 SEQ ID NO: 72 SEQ ID NO: 121 SEQ ID NO: 170IgG-Fc-24 SEQ ID NO: 24 SEQ ID NO: 73 SEQ ID NO: 122 SEQ ID NO: 171IgG-Fc-25 SEQ ID NO: 25 SEQ ID NO: 74 SEQ ID NO: 123 SEQ ID NO: 172IgG-Fc-26 SEQ ID NO: 26 SEQ ID NO: 75 SEQ ID NO: 124 SEQ ID NO: 173IgG-Fc-27 SEQ ID NO: 27 SEQ ID NO: 76 SEQ ID NO: 125 SEQ ID NO: 174IgG-Fc-28 SEQ ID NO: 28 SEQ ID NO: 77 SEQ ID NO: 126 SEQ ID NO: 175IgG-Fc-29 SEQ ID NO: 29 SEQ ID NO: 78 SEQ ID NO: 127 SEQ ID NO: 176IgG-Fc-30 SEQ ID NO: 30 SEQ ID NO: 79 SEQ ID NO: 128 SEQ ID NO: 177IgG-Fc-31 SEQ ID NO: 31 SEQ ID NO: 80 SEQ ID NO: 129 SEQ ID NO: 178IgG-Fc-32 SEQ ID NO: 32 SEQ ID NO: 81 SEQ ID NO: 130 SEQ ID NO: 179IgG-Fc-33 SEQ ID NO: 33 SEQ ID NO: 82 SEQ ID NO: 131 SEQ ID NO: 180IgG-Fc-34 SEQ ID NO: 34 SEQ ID NO: 83 SEQ ID NO: 132 SEQ ID NO: 181IgG-Fc-35 SEQ ID NO: 35 SEQ ID NO: 84 SEQ ID NO: 133 SEQ ID NO: 182IgG-Fc-36 SEQ ID NO: 36 SEQ ID NO: 85 SEQ ID NO: 134 SEQ ID NO: 183IgG-Fc-37 SEQ ID NO: 37 SEQ ID NO: 86 SEQ ID NO: 135 SEQ ID NO: 184IgG-Fc-38 SEQ ID NO: 38 SEQ ID NO: 87 SEQ ID NO: 136 SEQ ID NO: 185IgG-Fc-39 SEQ ID NO: 39 SEQ ID NO: 88 SEQ ID NO: 137 SEQ ID NO: 186IgG-Fc-40 SEQ ID NO: 40 SEQ ID NO: 89 SEQ ID NO: 138 SEQ ID NO: 187IgG-Fc-41 SEQ ID NO: 41 SEQ ID NO: 90 SEQ ID NO: 139 SEQ ID NO: 188IgG-Fc-42 SEQ ID NO: 42 SEQ ID NO: 91 SEQ ID NO: 140 SEQ ID NO: 189IgG-Fc-43 SEQ ID NO: 43 SEQ ID NO: 92 SEQ ID NO: 141 SEQ ID NO: 190IgG-Fc-44 SEQ ID NO: 44 SEQ ID NO: 93 SEQ ID NO: 142 SEQ ID NO: 191IgG-Fc-45 SEQ ID NO: 45 SEQ ID NO: 94 SEQ ID NO: 143 SEQ ID NO: 192IgG-Fc-46 SEQ ID NO: 46 SEQ ID NO: 95 SEQ ID NO: 144 SEQ ID NO: 193IgG-Fc-47 SEQ ID NO: 47 SEQ ID NO: 96 SEQ ID NO: 145 SEQ ID NO: 194IgG-Fc-48 SEQ ID NO: 48 SEQ ID NO: 97 SEQ ID NO: 146 SEQ ID NO: 195IgG-Fc-49 SEQ ID NO: 49 SEQ ID NO: 98 SEQ ID NO: 147 SEQ ID NO: 196

Example 1.1 Production of IgG-Fc Domain Binding VHH Fragments

The IgG-binding proteins of the invention were produced in yeast usingstrains and expression-constructs as described by van de Laar, et al.,(2007, Biotechnology and Bioengineering, Vol. 96, No. 3: 483-494).Production of IgG-binding proteins was performed in standard bioreactorswith a working volume of between 10 and 10,000 litres. Dissolved oxygen(Ingold DO2 electrode, Mettler-Toledo) was controlled by automaticadjustment of the impeller speed. The pH (Mettler-Toledo Inpro 3100 gelelectrode or Broadley James F635 gel electrode) was controlled usingphosphoric acid and ammoniac gas or ammonia solution. Foaming wasdetected by a foam level sensor (Thermo Russell) and controlled by 5-10%Struktol J673 addition. Temperature (PT100 electrode) was controlled viaa cooling jacket and heating jacket. The offgas (Prima 600 massspectrophotometer, VG gas analysis systems) analysed the ethanolconcentration, rO2 and rCO2. Adding 3%-8% full-grown inoculum startedthe batch phase (30° C., 0.3-0.4 VVM air, DO2 minimum 30%, pH 5.0). Theethanol fermentations were automatically started when the ethanolconcentration in offgas was declining in batch phase. The feed wasapplied according to a pulsed feed profile to maintain the ethanol levelwithin the demanded margins. The feed phases were performed at 21° C.and 0.7-1.1 VVM air. During the ethanol fermentations the DO2 decreasedto 0% and accumulated ethanol was further controlled by a pulsed feedprofile. Feed phase was stopped when the ethanol feed was depleted. Thebroth was chilled to a temperature between 5-10° C. until furtherprocessing, including removal of spent biomass removal.

Typical fermentation parameters include a temperature of 20-31° C., a pHof 4.7-5.8, product formed: 1000-1500 mg/l cell free broth, fermentationtime of 115-120 h and cell dry weight (at the end of fermentation):95-115 g/kg.

Example 1.2 Expression Levels of IgG-Fc Domain Binding VHH Fragments inFermentation

Expression levels of IgG-Fc binding VHH fragments in ethanol fedfermentations as described in example 1.1 were determined using aquantitative HPLC assay based on affinity chromatography columns Sampleswere loaded onto an IgG coupled affinity column After washout of unboundsample, bound IgG-Fc VHH fragment was eluted at low pH. The area of theeluted peak was determined by peak integration. Based on this peak area,the VHH fragment concentration in a sample was calculated using astandard curve.

End of Fermentation (EoF) samples at different fermentation volumes wereanalyzed on VHH fragment expression. An overview of VHH fragmentexpression levels at different fermentation volumes is presented inTable 6.

TABLE 6 Overview VHH fragment expression levels at differentfermentation volumes Production VHH fragment Batch id Fermentationvolume (g/l)* IgG-Fc-1 206024 10 m³ 1.13 206025 10 m³ 1.18 IgG-Fc-10204005 200 L 1.35 206002 10 m³ 1.58 IgG-Fc-16 206094 200 L 3.37*concentration is in g of VHH fragment per litre of supernatant at EoF

Example 2.1 ELISA and Biacore Analysis

For binding analysis in ELISA, Nunc Maxisorp binding plates were coatedwith antibody antigens of different species and subsequently blockedwith 2% (w/v) gelatin in PBS. Bound VHH fragments were detected byeither a mouse anti-His mAb in combination with a polyclonalgoat-anti-mouse-HRP conjugate (Bio-Rad, 172-1011) or a polyclonal rabbitanti-llama-VHH serum in combination with a polyclonal swine-anti-rabbitIgG-HPO conjugate (Dako, P217).

Binding analysis using surface plasmon resonance analysis (SPR) wereperformed on a BiaCore 3000. For this purpose, antibody antigens wereimmobilised onto the surface of a CM5 sensor chip and subsequentlyincubated with anti IgG-Fc VHH fragments in HBS-EP buffer (0.01 M HEPES,pH 7.4; 0.15 M NaCl; 3 mM EDTA; 0.005% Surfactant P20). Binding wasallowed for 1 minute at 5 μl/min followed by a dissociation step of 2.5minutes at 5 μl/min. Binding signals (Resonance Units) were compared tobackground signals measured with HBS-EP buffer only.

No discrepancy was found between ELISA—and Biacore measurements. Anoverview of the specificity of the tested anti IgG-Fc VHH fragments isgiven in Table 7. For comparison, the relative reactivity of Protein Aand G towards different IgG species is given in Table 8.

TABLE 7 Binding specificity of anti IgG-Fc VHH fragments (ELISA). IgG-Fcseq IgG Fc domain species no binding to IgG-Fc from 1-49 mammalian 1 to25, 31 to 36, 38, 43, 44 human mouse, bovine 1 to 25, 33, 34, 38, 44human (all subclasses) mouse, bovine, goat 1 to 15 human (allsubclasses) mouse, bovine, rat, syrian hamster, guinea pig, dog, cat,goat, sheep 1 to 9 human (all subclasses), mouse, bovine, rat, syrianHuman IgG recovery of at hamster, guinea pig, dog, cat, least 99% (0.1Mglycine, pH goat, sheep 3.0) 10 to 15 human (all subclasses), mouse,bovine, rat, syrian binding affinity at least 4 · 10⁹M⁻¹; hamster,guinea pig, dog, cat, expression level in yeast goat, sheep at least 1.2g/l 16, 26 to 30, 37, 42, 47 to 49 human, mouse, bovine (at least twospecies) 16, 28, 29, 30, 37, 42, 47 to 49 human, bovine, mouse, rat,rabbit, dog, cat, swine, sheep, monkey (chimpanzee, rhesus), donkey,horse 16, 28, 29, 30, 48 human, bovine, mouse, rat, rabbit, dog, cat,swine, sheep, monkey (chimpanzee, rhesus), donkey, horse, goat, syrianhamster, guinea pig

Anti IgG-Fc VHH fragments 36, 38-41, 43-46 show binding in ELISA tohuman IgG-Fc domains, however, no further analysis on other IgG specieswere performed.

Example 2.2 Biacore Analysis of IgG-Fc Domain Binding VHH FragmentIgG-Fc-16

Broad binding reactivity of VHH fragment IgG-Fc-16 was determined usingsurface plasmon resonance analysis (SPR) on a BiaCore 3000. For thispurpose, purified VHH fragment IgG-Fc-16 was immobilised onto thesurface of a CM5 sensor chip and subsequently incubated with purifiedIgG antibodies (50 μg/ml) from different species in HBS-EP buffer.Binding was allowed for 1 minute at 5 μl/min followed by a dissociationstep of 2.5 minutes at 5 μl/min. Binding signals (Resonance Units) werecompared to background signals measured with HBS-EP buffer only. Resultsare summarised in Table 8. For comparison, the relative reactivity ofProtein A and G towards different IgG species is also given.

TABLE 8 Broad species reactivity of IgG-Fc domain binding VHH fragmentIgG-Fc-16 in Biacore Binding signal on Binding reactivity of IgG speciesIgG-Fc-16 (RU) Protein A/Protein G HuIgG 1112.6 +++/+++ Human IgG, Fcfragment 806.8 +++/+++ Human IgG, Fab fragment −2.8 +/+ HuIgG1 1569.0+++/+++ HuIgG2 794.7 +++/+++ HuIgG3 499.0 −−/+++ HuIgG4 710.5 +++/+++Rat IgG 107.7 +/++ Rat IgG1a 431.1 −−/+ Rat IgG2a 443.9 −−/+++ Rat IgG2b512.4 −−/+ Rat IgG2c 2134.5 ++/++ Rabbit IgG 799.6 +++/+++ Sheep IgG845.5 +/++ Bovine IgG 308.8 +/+++ Bovine IgG, Fc fragment 172.1 BovineIgG, Fab fragment −1.7 Mouse IgG 468.1 ++/++ Mouse IgG 1 1612.6 +/++Mouse IgG2a 426.6 +++/+++ Dog IgG 489.9 +++/+ Goat IgG. 257.1 +/++Syrian Hamster IgG 670.2 +/++ Swine IgG 810.7 +++/++ Cat IgG 409.5 +++/+Donkey IgG 637.6 Guinea IgG 576.9 +++/+ Foetal Calf IgG 260.0 +/+++Newborn Calf IgG 238.8 +/+++ Chicken IgG −3.1 −−/+ Horse IgG 429.6 +/+++Buffer −3.0 +++; ++; +; −−: strong binding; moderate binding; weakbinding; no binding, respectively

Note that the “+” value as given in Table 8 for binding of Protein A tohuman IgG Fab fragments only relates to human Fab fragments comprising aVH domain belonging to the human V_(H)-III family. Observed bindingreactivity of Protein G towards human IgG Fab fragments occurs throughbinding to an epitope present on the C_(H)1 domain of human IgGantibodies. For both Protein A and -G, however, the binding strengthtowards these Fab related epitopes is less compared to the epitopespresent on the Fc domain of IgG antibodies. As shown in Table 8, VHHfragment IgG-Fc-16, does not co-bind to any epitope present on IgG Fabfragments.

Example 2.3 Binding Affinity Measurements on Biacore

Binding affinity constants of anti IgG-Fc VHH fragments were determinedusing surface plasmon resonance analysis (SPR) on a BiaCore 3000. Forthis purpose, purified VHH fragments were immobilised onto the surfaceof a CM5 sensor chip and subsequently incubated with differentconcentrations of purified IgG antibodies in HBS-EP buffer. Binding wasallowed for 3 minutes at 30 μl/min followed by a dissociation step of 15minutes at 30 μl/min Binding curves were fitted according to a 1:1Langmuir binding model using Biacore software. An overview of thecalculated affinity data is given in Table 9.

TABLE 9 Biacore affinity data of anti IgG-Fc VHH fragments k ass k dissKA KD VHH Antigen (1/Ms) (1/s) (1/M) (M) IgG-Fc-1 Human- 8.24 × 10⁴ 1.76× 10⁻⁴ 4.67 × 10⁸ 2.14 × 10⁻⁹ IgG IgG-Fc-1 Human- 3.61 × 10⁵ 2.08 × 10⁻⁴1.74 × 10⁹ 5.75 × 10⁻¹⁰ IgG1 IgG-Fc-1 Human- 1.22 × 10⁵ 2.12 × 10⁻⁴ 5.76× 10⁸ 1.73 × 10⁻⁹ IgG2 IgG-Fc-1 Human- 5.69 × 10³ 2.72 × 10⁻⁴ 2.09 × 10⁷4.78 × 10⁻⁸ IgG3 IgG-Fc-1 Human- 1.96 × 10⁵ 5.91 × 10⁻⁵ 3.32 × 10⁹ 3.02× 10⁻¹⁰ IgG4 IgG-Fc-10 Human- 3.96 × 10⁵ 1.29 × 10⁻³ 3.07 × 10⁸ 3.25 ×10⁻⁹ IgG IgG-Fc-10 Human- 2.49 × 10⁵ 6.54 × 10⁻⁴ 3.81 × 10⁸ 2.62 × 10⁻⁹IgG1 IgG-Fc-10 Human- 1.22 × 10⁵ 8.89 × 10⁻⁴ 1.37 × 10⁸ 7.29 × 10⁻⁹ IgG2IgG-Fc-10 Human- 1.56 × 10⁵ 1.40 × 10⁻³ 1.11 × 10⁸ 8.99 × 10⁻⁹ IgG3IgG-Fc-10 Human- 1.00 × 10⁵ 1.23 × 10⁻³ 8.16 × 10⁷ 1.23 × 10⁻⁸ IgG4IgG-Fc-16 Human- 3.53 × 10⁴ 4.62 × 10⁻⁴ 7.65 × 10⁷ 1.31 × 10⁻⁸ IgGIgG-Fc-16 Human- 3.10 × 10⁴ 2.11 × 10⁻⁴ 1.47 × 10⁸ 6.81 × 10⁻⁹ IgG1IgG-Fc-16 Human- 2.19 × 10⁴ 3.55 × 10⁻⁴ 6.17 × 10⁷ 1.62 × 10⁻⁸ IgG2IgG-Fc-16 Human- 3.98 × 10⁴ 8.49 × 10⁻⁴ 4.69 × 10⁷ 2.13 × 10⁻⁸ IgG3IgG-Fc-16 Human- 6.29 × 10⁴ 1.07 × 10⁻⁴ 5.89 × 10⁸ 1.70 × 10⁻⁹ IgG4IgG-Fc-16 Human- 3.10 × 10⁴ 2.11 × 10⁻⁴ 1.47 × 10⁸ 6.81 × 10⁻⁹ IgG1IgG-Fc-16 Bovine- 1.38 × 10⁵ 3.86 × 10⁻⁴ 3.59 × 10⁸ 2.79 × 10⁻⁹ IgGIgG-Fc-16 Mouse- 2.35 × 10⁴ 1.98 × 10⁻⁴ 1.19 × 10⁸ 8.41 × 10⁻⁹ IgG

Example 3.1 Chromatography Testing

Purified anti IgG-Fc VHH fragments were dialysed to NHS coupling bufferand coupled to NHS activated sepharose 4B Fast Flow according to thesuppliers protocol (GEHC) and as described in WO2006/059904. Columnswere made of the coupled antibody matrix using HR 5/5 columns (GEHC). Acolumn volume of 400 μl was used. All the chromatography experimentswere performed on an Akta explorer 100. IgG samples were loaded in PBSpH 7.4, and eluted using e.g. PBS with addition of 8 M HCl to yield pH2.1 or 0.1 M Glycine-HCl at pH 2 or 3. Protein detection was performedon line by monitoring the signal of OD₂₁₄ and OD₂₈₀. An overview of thebinding analysis of the tested anti IgG-Fc sepharose carriers inchromatography are given in Table 10.

TABLE 10 Binding of IgG on anti IgG-Fc sepharose carriers inchromatography VHH-Sepharose Human Mouse Bovine carrier IgG IgG IgGIgG-Fc-1 + − − IgG-Fc-10 + − − IgG-Fc-15 + − − IgG-Fc-24 − + −IgG-Fc-29 + − + IgG-Fc-16 + + + IgG-Fc-48 + + −

Example 3.1 Dynamic Binding Capacities of IgG-Fc Domain Binding VHHFragments

The dynamic binding capacity (DBC) of the IgG-Fc domain binding VHHfragments immobilized onto NHS sepharose was tested. 10 ml of 1.0 mg/mlhuman IgG in PBS pH 7.4 was loaded on a 400 μl column with a linear flowof 150 cm/h. After the washing with 10 column volumes PBS pH 7.4, thecolumn was eluted with 0.1 M glycine buffer pH 3.0. Based on integrationof the OD280 signal of the flow through and elution peak, the dynamicbinding capacity of the column was calculated (Table 11).

TABLE 11 Dynamic binding capacity of anti-IgG-Fc sepharose carriersDynamic binding capacity IgG-Fc binding VHH fragments (mg human IgG/mlmatrix) IgG-Fc-1 18.2 IgG-Fc-10 16.7 IgG-Fc-16 11.5

Example 3.2 Elution Profile of IgG-Fc Domain Binding VHH FragmentIgG-Fc-1 in Chromatography

Purified VHH fragment IgG-Fc-1 was dialysed to NHS coupling buffer andcoupled to NHS activated sepharose 4B Fast Flow according to thesuppliers protocol (GEHC) and as described in WO2006/059904. Columnswere made of the coupled antibody matrix using HR 5/5 columns (GEHC). Acolumn volume of 400 ml was used. For comparison a Protein A HiTrapcolumn (1 ml) was used. All the chromatography experiments wereperformed on an Akta explorer 100. IgG samples (1 mg/ml) were loaded (10ml on IgG-Fc-1 sepharose, 20 ml on Protein A HiTrap) in PBS pH 7 andeluted using the following types of first elution buffers:

-   -   0.2M Glycine in miliQ with different pH value's    -   0.1M Acetic Acid in miliQ with different pH value's    -   0.1M Citric Acid in miliQ with different pH value's

As a second elution buffer (regeneration) PBS, pH2 was used.

After sample loading (linear flow: 150 cm/hr) and washout of unboundsample (linear flow: 150 cm/hr; volume: 10 cv), the first elution(linear flow: 300 cm/hr; volume: 30 cv) is carried out with one of thefirst elution buffers at a specific pH (see table 12).

After re-equilibration of the column with binding buffer (linear flow:300 cm/hr; volume 30 cv), a second elution (regeneration) is carried outwith the second elution buffer (PBS, pH 2.0; linear flow: 300 cm/hr;volume 20 cv).

From the chromatograph, the two elution peaks are integrated, and therelation between the two was calculated. The data are presented in Table12.

TABLE 12 Elution profile of VHH fragment IgG-Fc-1 in chromatography incomparison with Protein A 0.2M Glycine Protein 0.1M Acetic Acid 0.1MCitric Acid pH IgG-Fc-1 A IgG-Fc-1 Protein A IgG-Fc-1 Protein A 2 100*100 100 100 100 100 3 100  100 99 99 99 99 4 98 96.5 85 72 92 84 4.5 8756 62 42 75 63 5 78 19 44 23 60 33 *percentage of the first elution peakin comparison with the total peak area of the first and second elutionpeak.

Example 3.3 Elution Profile of IgG-Fc Domain Binding VHH FragmentIgG-Fc-16 in Chromatography

Purified VHH fragment IgG-Fc-16 was dialysed to NHS coupling buffer andcoupled to NHS activated sepharose 4B Fast Flow according to thesuppliers protocol (GEHC) and as described in WO2006/059904. Columnswere made of the coupled antibody matrix using HR 5/5 columns (GEHC). Acolumn volume of 400 μl was used. All the chromatography experimentswere performed on an Akta explorer 100. IgG samples (1 mg/ml) wereloaded (10 ml on VHH sepharose) in PBS pH7 and eluted using thefollowing types of first elution buffers:

-   -   0.1M Glycine in miliQ with different pH value's    -   0.1M Arginine in miliQ with different pH value's

As a second elution buffer (regeneration) PBS, pH2 was used.

After sample loading (linear flow: 150 cm/hr) and washout of unboundsample (linear 150 cm/hr; volume: 10 cv), the first elution (linearflow: 300 cm/hr; volume: 30 cv) is carried out with one of the firstelution buffers at a specific pH (see table 13). After re-equilibrationof the column with binding buffer (linear flow: 300 cm/hr; volume 30cv), a second elution (regeneration) is carried out with the secondelution buffer (PBS, pH 2.0; linear flow: 300 cm/hr; volume 20 cv).

From the chromatograph, the two elution peaks are integrated, and therelation between the two was calculated. The data are presented in table13.

TABLE 13 Elution profile of VHH fragment IgG-Fc-16 in chromatographyFirst elution buffer Elution (%) 0.1M Arginine pH 3.0 100* 0.1M GlycinepH 3.0 100  0.1M Arginine pH 4.0 37 0.1M Glycine pH 4.0 86 *percentageof the first elution peak in comparison with the total peak area of thefirst and second elution peak.

Example 3.4 Caustic Stability of VHH Fragment IgG-Fc-1 in Chromatography

Purified VHH fragment IgG-Fc-1 was dialysed to NHS coupling buffer andcoupled to NHS activated agarose according to the suppliers protocol.Columns were made of the coupled antibody matrix using HR 5/5 columns(GEHC). 10 ml of 1.0 mg/ml human IgG in PBS pH 7.4 was loaded on a 400n1 column with a linear flow of 150 cm/h. After the washing with 10column volumes PBS pH 7.4, the column was eluted with PBS adjusted to pH2.1 with 8 M HCl. Based on integration of the OD280 signal of the flowthrough and elution peak, the dynamic binding capacity (DBC) of thecolumn was calculated. The column was then incubated with 0.1 M or 0.2 MNaOH for 15 minutes at a linear flow of 150 cm/hr followed byequilibration with 10 column volumes of PBS pH 7.4. After each cycle thedynamic binding capacity was determined as described above. The residualDBC's after cycles with 0.1 M and 0.2 M NaOH are presented in FIG. 1.

No loss in DBC was found after more than 100 cycles with 0.1 M NaOH.When incubated with 0.2 M NaOH, a residual DBC of more than 90% wasfound after 40 cycles.

1-44. (canceled)
 45. An antigen-binding protein comprising an amino acidsequence that comprises 4 framework regions, FRI to FR4, and 3complementarity determining regions, CDR1 to CDR3, that are operablylinked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein: a) the CDR1has an amino acid sequence selected from the group consisting of SEQ IDNo's: 1-49 or an amino acid sequence that differs from SEQ ID No's: 1-49in one or two of the amino acid residues; b) the CDR2 has an amino acidsequence having at least 80% sequence identity with an amino acidsequence selected from the group consisting of SEQ ID No's: 50-98; and,c) the CDR3 is an amino acid sequence having at least 80% sequenceidentity with an amino acid sequence selected from the group consistingof SEQ ID No's: 99-147; and, wherein each of the framework regions hasat least 50% amino acid identity with the framework amino acid sequenceof any one of SEQ ID No's: 148-196, and wherein the antigen-bindingprotein specifically binds to the Fc domain of a mammalian IgG molecule.46. The antigen-binding protein according to claim 45, wherein: a) theCDR1 has an amino acid sequence selected from the group consisting ofSEQ ID No's: 1-15 and amino acid sequences that differs from SEQ IDNo's: 1-15 in no more than 4 amino acid residues; b) the CDR2 has anamino acid sequence selected from the group consisting of SEQ ID No's:50-64 and an amino acid sequences that differs from SEQ ID No's: 50-64in no more than 6 amino acid residues; and, c) the CDR3 has an aminoacid sequence selected from the group consisting of SEQ ID No's: 99-113and an amino acid sequences that differs from SEQ ID No's: 99-113 in nomore than 6 amino acid residues.
 47. The antigen-binding proteinaccording to claim 46, wherein the antigen binding protein has one ormore properties selected from the group consisting of: a) theantigen-binding protein binds the human IgG molecule with a bindingaffinity of at least 3 nM as analyzed by surface plasmon resonanceanalysis (SPR) using polyclonal human IgG; and, b) the antigen-bindingprotein is obtainable by expression in yeast at an expression level ofat least 1.2 g/L of yeast culture.
 48. A host cell comprising a nucleicacid encoding the amino acid sequence of claim
 45. 49. The host cellaccording to claim 48 wherein the host cell is a yeast.
 50. A method forproducing the antigen-binding protein of claim 45, the method comprisingthe steps of: a) culturing a host cell according to claim 48 underconditions conducive to expression of the antigen-binding protein; andoptionally, b) purifying the antigen-binding protein from at least oneof the host cell and the culture medium.
 51. A composition comprisingthe antigen-binding protein of claim
 45. 52. An immunoadsorbent materialcomprising the antigen-binding protein of claim
 45. 53. Theimmunoadsorbent material of claim 52, wherein the antigen-bindingprotein is immobilized onto a carrier, wherein preferably theantigen-binding protein is immobilized onto the carrier by a covalentlink.
 54. A method for the purification of a mammalian IgG molecule, themethod comprising the steps of: a) bringing a sample comprising themammalian IgG molecule in contact with the immunoadsorbent material ofclaim 52 under conditions that allow binding of the mammalian IgGmolecule to the immunoadsorbent material; b) optionally, performing awashing step; c) eluting the bound mammalian IgG molecule underconditions that decrease the affinity between the mammalian IgG moleculeand the immunoadsorbent material; and, d) optionally, further processingmammalian IgG molecule.
 55. A nucleic acid comprising a nucleotidesequence encoding an antigen-binding protein according to claim
 45. 56.The nucleic acid according to claim 55, wherein the nucleotide sequenceis operably linked to a promoter and optionally other regulatoryelements.